A CD8 antibody-modified anti-TIM-3 lipid nanoparticle, preparation method and application thereof

Abstract

The present invention provides a CD8 antibody-modified anti-TIM-3 lipid nanoparticle, a preparation method and its application, which belongs to the technical field of medical preparations, wherein the lipid nanoparticle contains a chimeric antigen receptor; the chimeric antigen receptor includes a single-chain antibody TIM-3; the nucleic acid artificial sequence of the single-chain antibody TIM-3 is shown in SEQ ID NO.5. The present application performs sequence optimization on the TIM-3 target, prepares a CAR structure connecting a dual stimulatory factor and a suicide gene, and constructs an anti-TIM-3 nanolipid particle modified by a CD8 antibody, which is delivered to cells, can effectively capture CD8+T cells in the body, produce CAR-T cells, eliminate the complex process of in vitro preparation, and improve the activation degree of T cells while efficiently recognizing antigens, exerting a highly effective anti-tumor effect, significantly prolonging the survival of breast cancer mice, and inhibiting tumor growth.

Description

Technical Field

[0001] The present invention relates to a CD8 antibody-modified anti-TIM-3 lipid nanoparticle and application thereof, belonging to the technical field of medical preparations. Background Art

[0002] T cell immunoglobulin and mucin domain molecule 3 (abbreviated as TIM-3), also known as hepatitis A virus cell receptor 2, belongs to the Tim gene family. The TIM-3 target has important potential in tumor immunotherapy because it can bind to a variety of ligands, such as galectin-9 (Gal-9), carcinoembryonic antigen cell adhesion molecule-1 (CEACAM-1), high mobility group protein B1 (HMGB1) and phosphatidylserine (PS). The interaction between these ligands and TIM-3 plays a key role in regulating T cell immunity. The inhibition of TIM-3 can enhance the anti-tumor immune response. Therefore, many pharmaceutical companies are developing immunotherapy drugs targeting TIM-3.

[0003] Lipid nanoparticles (LNPs) are a highly efficient drug delivery system, widely used in the encapsulation and delivery of nucleic acid drugs, small molecule compounds, etc. Its main components include ionizable lipids, cholesterol, auxiliary lipids and polyethylene glycol (PEG) lipids, which work together to protect nucleic acid drugs from degradation, improve the solubility and efficacy of drugs, and achieve targeted drug delivery. They show broad application prospects in the fields of nucleic acid drug delivery, cancer treatment and vaccine development.

[0004] At present, clinical research targeting TIM-3 is mainly focused on the treatment of solid tumors such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and non-small cell lung cancer, and most TIM-3 drugs are in the clinical stage. CAR cell therapy targeting TIM-3 is exploring its therapeutic potential in clinical trials. A clinical trial (NCT06125652) is evaluating the safety and efficacy of anti-TIM-3 / CD123 CAR-T cells in the treatment of relapsed and refractory acute myeloid leukemia, but TIM-3 antibodies have shown general single-drug effects in preclinical and clinical stages, and even when used in combination with PD-1 antibodies, the effect is lower than expected.

[0005] CD8 antibody-modified anti-TIM-3 lipid nanoparticles have not been reported in the prior art. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a CD8 antibody-modified anti-TIM-3 lipid nanoparticle, a preparation method and an application thereof, to achieve the following invention objectives: after the CD8 antibody-modified anti-TIM-3 lipid nanoparticles are transfected with CD8+T cells in vitro, the killing rate of the target cell MCF-7 is improved; the CD8 antibody-modified anti-TIM-3 lipid nanoparticles can capture CD8+T cells in vivo, produce CAR-T cells, and improve the anti-tumor effect.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A CD8 antibody-modified anti-TIM-3 lipid nanoparticle, wherein the lipid nanoparticle contains a chimeric antigen receptor; the chimeric antigen receptor includes a single-chain antibody TIM-3; the artificial nucleic acid sequence of the single-chain antibody TIM-3 is shown in SEQ ID NO.5.

[0009] The chimeric antigen receptor is obtained by sequentially connecting the following modules: a guide, a single-chain antibody TIM-3, a CD8 Hinge region, a CD28 transmembrane region, a CD28-4-1BB co-stimulatory region, a CD3ζ intracellular region, a self-cleavage region T2A, and a suicide gene.

[0010] The artificial nucleic acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.2.

[0011] The heavy chain amino acid sequence of the CD8 antibody is shown in SEQ ID NO.12, and the light chain amino acid sequence is shown in SEQ ID NO.13.

[0012] The preparation method of the CD8 antibody-modified anti-TIM-3 lipid nanoparticles is to construct a chimeric antigen receptor to obtain a recombinant plasmid, encapsulate it with LNP, and then couple it with CD8 antibody to obtain CD8 antibody-modified anti-TIM-3 lipid nanoparticles.

[0013] The lipid nanoparticles are used in the preparation of anti-breast cancer drugs.

[0014] Compared with the prior art, the present invention achieves the following beneficial effects:

[0015] The present application optimizes the sequence of the TIM-3 target, prepares a CAR structure connected to a dual stimulatory factor and a suicide gene, and constructs anti-TIM-3 nanolipid particles modified with CD8 antibodies, which are delivered into cells. The CAR-T cells can effectively capture CD8+T cells in the body and produce CAR-T cells, eliminating the complex process of in vitro preparation. While efficiently recognizing antigens, the activation level of T cells is improved, and an efficient anti-tumor effect is exerted, which significantly prolongs the survival of breast cancer mice and inhibits tumor growth.

[0016] The anti-TIM-3 nanolipid particles modified with CD8 antibodies prepared by the present invention are used to transfect CD8+T cells in vitro to obtain CAR-T cells, and the killing rate of the target cell MCF-7 reaches 67.9%. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a flow cytometric graph of the infection rate of AntiCD8-LNP-TIM-3-1 on activated CD8+T cells;

[0018] Figure 2 It is a flow cytometric graph of the infection rate of AntiCD8-LNP-TIM-3-2 on activated CD8+T cells;

[0019] Figure 3 It is a bar graph showing the killing rate of MCF-7 cells by CAR-T-1 cells, CAR-T-2 cells, and activated CD8+T cells;

[0020] Figure 4 This is a graph showing the determination of the survival period of mice in Example 6;

[0021] Figure 5 This is a graph showing the mouse tumor growth curve in Example 6. DETAILED DESCRIPTION

[0022] Example 1 Design of target gene and construction of recombinant plasmid

[0023] The modules of the CAR structure and their artificial nucleic acid sequences are as follows:

[0024] (1) a guide, the artificial nucleic acid sequence of which is shown as SEQ ID NO.3 in the sequence listing;

[0025] (2) single-chain antibody TIM-3, the artificial nucleic acid sequence before optimization is shown in SEQ ID NO.4 in the sequence listing; the artificial nucleic acid sequence after optimization is shown in SEQ ID NO.5 in the sequence listing;

[0026] (3) CD8 Hinge region, the artificial nucleic acid sequence of which is shown in SEQ ID NO.6 in the sequence listing;

[0027] (4) CD28 transmembrane region, whose artificial nucleic acid sequence is shown as SEQ ID NO.7 in the sequence list;

[0028] (5) CD28-4-1BB co-stimulatory region, whose artificial nucleic acid sequence is shown in SEQ ID NO.8 in the sequence list;

[0029] (6) CD3ζ intracellular region, the artificial nucleic acid sequence of which is shown in SEQ ID NO.9 in the sequence listing;

[0030] (7) self-cleavage region T2A, whose artificial nucleic acid sequence is shown in SEQ ID NO.10 in the sequence listing;

[0031] (8) A suicide gene, whose artificial nucleic acid sequence is shown as SEQ ID NO.11 in the sequence listing.

[0032] The artificial nucleic acid sequences of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11 were connected in sequence, and the connected sequence is shown in SEQ ID NO.1 in the sequence table. Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the entire expression frame and insert it into the pUC57 vector, named pUC-CAR-1. The pUC-CAR vector and the PVAX1 vector were double-digested with Fast Digest BamH I (purchased from ThermoFisher) and Fast Digest Not I (purchased from ThermoFisher), and the linearized DNA fragments were obtained by gel cutting and recovery, and the PVAX1-CAR-TIM-3-1 expression vector was formed by overnight connection at 16°C. The above-mentioned PVAX1-CAR-TIM-3-1 expression vector was transformed into E. coli (DH5α), and the positive clones were picked for PCR identification. Then the plasmid was extracted and sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing. If the sequencing identification was correct, PVAX1-CAR-TIM-3-1 was successfully constructed. The PVAX1-CAR-TIM-3-1 recombinant plasmid was extracted from the positive clones, diluted to 2 μg / μL, and stored at -80°C for use.

[0033] Using the same method, the artificial nucleic acid sequences of SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11 were connected in sequence. The connected sequence is shown in SEQ ID NO.2 in the sequence table. The PVAX1-CAR-TIM-3-2 expression vector was successfully constructed according to the above method, and the PVAX1-CAR-TIM-3-2 recombinant plasmid was extracted from the positive clone, diluted to 2 μg / μL, and stored at -80°C for use.

[0034] Example 2 Preparation of LNPs carrying recombinant plasmids

[0035] (1) Take 10.9 μL of the PVAX1-CAR-TIM-3-1 recombinant plasmid prepared in Example 1 (concentration 2.0 μg / μL), dilute to 300 μL using a citric acid buffer with a pH of 4 and a concentration of 10 mM to obtain a recombinant plasmid mixture;

[0036] (2) Dissolve 2.18 mg of ionizable lipid DLin-MC3-DMA, 1.00 mg of cholesterol, 0.27 mg of DMG-PEG2000, 0.54 mg of DSPC, and 0.1 mg of DSPE-PEG-maleimide in 1 mL of ethanol to obtain a mixed lipid solution;

[0037] (3) A recombinant plasmid mixture and a mixed lipid solution were added to a microfluidic device, wherein the mass ratio of DNA in the recombinant plasmid mixture to ionizable lipid DLin-MC3-DMA in the mixed lipid solution was 1:10. LNP nanoparticles encapsulating the recombinant plasmid were prepared by a microfluidic method.

[0038] The obtained LNP nanoparticles were dialyzed in a PBS solution (pH 7.4) for 24 h, concentrated using an Amicon ultracentrifugal filter, and filtered through a 0.22 μm filter membrane twice to obtain LNPs containing the recombinant plasmid, named LNP-TIM-3-1.

[0039] The LNP containing the recombinant plasmid PVAX1-CAR-TIM-3-2 was prepared by the same method as above and named LNP-TIM-3-2.

[0040] The particle sizes of LNP-TIM-3-1 and LNP-TIM-3-2 were measured using a dynamic light scattering particle size potential meter, and the encapsulation efficiency was measured using the Quant-iT 1X dsDNA kit (Invitrogen).

[0041] The results showed that the encapsulation efficiency of LNP-TIM-3-1 was 85.3±4.36%, which was a high encapsulation efficiency, and the particle size was 105.6±5.0nm, which was uniform; the encapsulation efficiency of LNP-TIM-3-2 was 87.2±4.17%, which was a high encapsulation efficiency, and the particle size was 108.2±5.8nm, which was uniform.

[0042] Example 3 Coupling CD8 Antibody

[0043] The heavy chain amino acid sequence of the CD8 antibody is shown in SEQ ID NO.12 in the sequence list, and the light chain amino acid sequence is shown in SEQ ID NO.13 in the sequence list. The CD8 antibody is prepared according to conventional methods.

[0044] The CD8 antibody was reduced in PBS containing 1 mM DTT and 5 mM EDTA at room temperature for 1 h, and then DTT was removed by buffer exchange into PBS containing 5 mM EDTA using a 7KZeba desalting spin column (purchased from ThermoFisher Scientific). Immediately after buffer exchange, the reduced CD8 antibody was added to LNP-TIM-3-1 (0.67 mg of CD8 antibody was added per ml of LNP-TIM-3-1), and the mixture was incubated at room temperature with gentle shaking for 2 hours, and then incubated at 4°C overnight. On the next day, using PBS as the mobile phase, CD8 antibody-coupled LNP-TIM-3-1 was separated from free antibodies on a sepharose gel CL-4B filter column and concentrated to the initial volume of LNP-TIM-3-1 using a 100K Millipore ultrafiltration centrifuge tube (purchased from Solebol) to obtain LNP-TIM-3-1 coupled to CD8 antibody, named AntiCD8-LNP-TIM-3-1.

[0045] LNP-TIM-3-2 was coupled to CD8 antibody in the same way to obtain AntiCD8-LNP-TIM-3-2.

[0046] The AntiCD8-LNP-TIM-3-1 and AntiCD8-LNP-TIM-3-2 obtained by lysis with Triton X-100 were respectively used to measure the amount of DNA using the Qubit™ dsDNA BR Assay Kit. The results showed that the concentration of DNA in AntiCD8-LNP-TIM-3-1 was 52 ng / μL, and the concentration of DNA in AntiCD8-LNP-TIM-3-2 was 54 ng / μL. The concentration of DNA in AntiCD8-LNP-TIM-3-1 and AntiCD8-LNP-TIM-3-2 was adjusted to 50 ng / μL and stored for later use.

[0047] Example 4 Two AntiCD8-LNP-TIM-3 transfected CD8+T cells

[0048] (1) Preparation of activated CD8+ T cells

[0049] 75 mL of the patient's autologous peripheral blood was collected and peripheral blood mononuclear cells were separated using Ficoll-Paque lymphocyte separation medium. The separated cells were sorted out with CD8+ sorting reagent provided by BD to sort out CD8+ T cells. The cells were counted and counted according to 1×10 6The cells were inoculated at 100 cells / mL, KBM551 cell culture medium (purchased from Corning, catalog number: 88-551-CM) containing a final concentration of 1500 IU / mL IL-2 was added, and then the same number of CD3CD28 magnetic beads as the cells were added to activate T cells. After 24 hours of activation, the magnetic beads were removed to obtain activated CD8+ T cells.

[0050] (2) Transfection of CD8+T cells

[0051] Adjust the number of activated CD8+ T cells to 1×10 7 0.1 mL of cell suspension was inoculated into a 6-well plate and placed at 37°C and 5% CO 2 , cultured overnight in a saturated humidity incubator, then added 100 ng of AntiCD8-LNP-TIM-3-1 prepared in Example 3, incubated at 37° C. for 48 h, and collected AntiCD8-LNP-TIM-3-1-CAR-T cells by centrifugation, referred to as CAR-T-1.

[0052] The same method was used to obtain AntiCD8-LNP-TIM-3-2-CAR-T cells, referred to as CAR-T-2.

[0053] The infection rate of AntiCD8-LNP-TIM-3 on activated CD8+T cells was detected by flow cytometry. Figure 1 and Figure 2 As shown, the infection rate of AntiCD8-LNP-TIM-3-1 on activated CD8+T cells was 58.7%, and the infection rate of AntiCD8-LNP-TIM-3-2 on activated CD8+T cells was 65.6%.

[0054] Example 5 In vitro anti-tumor experiment

[0055] (1) Target cells

[0056] Human breast cancer cell line MCF-7 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were used as target cells. MCF-7 cells frozen at -80°C were revived and inoculated in RPMI1640 medium containing 10% vol% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin at 37°C and 5% CO. 2 , culture in a saturated humidity incubator, and when the cell confluence reaches more than 80%, subculture is carried out; a total of 5 generations are cultured to fully activate the cells.

[0057] (2) Cell co-culture

[0058] Effector cells (1×10 5 / well) and target cells (1×10 5 / well) and were inoculated in a 96-well plate. The effector cells were the CAR-T-1 cells and CAR-T-2 cells prepared in Example 4, and the control group was the activated CD8+T cells prepared in Example 4.

[0059] Divided into three groups, the specific groups are as follows:

[0060] Experimental group A: CAR-T-1 cells and MCF-7 cells were co-cultured;

[0061] Experimental group B: CAR-T-2 cells and MCF-7 cells were co-cultured;

[0062] Control group: activated CD8+T cells co-cultured with MCF-7 cells;

[0063] Three replicates were performed in each group under 5% CO 2 , cultured in a 37°C incubator, added 20 mL of CCK-8 to each well after 24 hours, and continued to incubate for 2 hours. The OD value was read using a microplate reader at 450 nm. The cell killing rate was calculated as follows: killing rate / % = [1-(blank group OD value-effect cell group OD value) / blank group OD value] × 100%.

[0064] The results of the kill rate are as follows Figure 3 As shown in the figure, the killing rates of experimental group A and experimental group B were 42.5% and 67.9% respectively, and the killing rate of the control group was 14.8%. The killing rate of experimental group B was higher than that of experimental group A, indicating that the optimization of the ScFv (TIM-3) sequence had a significant effect on the tumor killing effect of the final prepared CAR-T cells.

[0065] Example 6 In vivo anti-tumor experiment

[0066] (1) Animal model preparation

[0067] After 6-8-week-old Balb / c nude mice were acclimated for one week, the MCF-7 cells in the logarithmic growth phase with good growth status were collected by centrifugation and resuspended in sterile saline to adjust the cell concentration to 1×10 7 / mL, 0.5mL of cell suspension was injected into the right shoulder blade of nude mice using a sterile syringe; the tumor size was then observed every day, and the long diameter (L) and short diameter (W) of the tumor were recorded. The tumor volume (V) was calculated as follows: Vtumor = (L×W 2 ) / 2, when the tumor volume reaches 200-300mm 3 The model was successfully used for subsequent experiments.

[0068] (2) Animal survival observation

[0069] The nude mice with successful modeling were randomly divided into 4 groups, with 12 mice in each group, namely:

[0070] Control group: injected with 200 μL normal saline;

[0071] Experimental group A: injection of 1×10 6 activated CD8+T cells;

[0072] Experimental group B: injection of 200 μL AntiCD8-LNP-TIM-3-1;

[0073] Experimental group C: injection of 200 μL AntiCD8-LNP-TIM-3-2;

[0074] The survival status of experimental animals was observed and recorded every day. Figure 4 As shown, the survival period of mice in experimental group A was much lower than that in experimental groups B and C, and the survival period of mice in experimental group C was longer than that in experimental group B, and the survival rate was higher, indicating that the anti-TIM-3 nanolipid particles prepared after ScFv (TIM-3) sequence optimization have significantly improved anti-tumor effects.

[0075] The AntiCD8-LNP-TIM-3-1 and AntiCD8-LNP-TIM-3-2 prepared in the present application can induce the production of CAR-T cells in vivo and can significantly prolong the survival of breast cancer animals.

[0076] (3) Tumor growth curve determination

[0077] Male C57BL6 mice aged 6-8 weeks (purchased from Nanjing Junke Biotechnology Co., Ltd.) were housed in an animal room (room temperature 23±2°C, humidity 50%±10%). MCF-7 cells in the logarithmic phase were collected and diluted to 1×10 in phosphate buffered saline (PBS). 6 Under sterile conditions, 0.2 mL of MCF-7 cell suspension was inoculated in the left armpit of the mouse and observed for two weeks. The appearance of hard nodules the size of rice grains in the armpit was considered as the standard for successful modeling.

[0078] C57BL6 breast cancer model mice (the size of the subcutaneous tumor tissue block measured with a vernier caliper was 90-100 mm 3 ) were randomly divided into 5 groups, 10 in each group, and the injection treatment experiment began:

[0079] a. Control group, the same volume of normal saline was injected into the tail vein;

[0080] b. Treatment group: 1×10 6 Cells / activated CD8+T cells;

[0081] c. Treatment group 2, 200 μL AntiCD8-LNP-TIM-3-1 / mouse injected into the tail vein;

[0082] d. Treatment group 3, tail vein injection of 200 μL AntiCD8-LNP-TIM-3-2 / mouse;

[0083] e. Four groups were treated with tail vein injection of 200 μL AntiCD8-LNP-TIM-3-2 / mouse, followed by an injection of rituximab 24 hours later (rituximab was also injected 24 hours after the second immunization).

[0084] The above-mentioned groups of mice were immunized once a week for two consecutive weeks. The day of the first immunization was recorded as day 0. The size of the subcutaneous tumor tissue blocks of mice in each treatment group was measured by vernier caliper every three days, and the tumor growth curve was drawn with the mean tumor volume. After 5 weeks, the mice were dissected, and the tumor tissues were embedded in paraffin and sectioned.

[0085] Tumor volume growth results Figure 5 As shown, on the 30th day after the first immunization, the average tumor volume of the control group mice was 3980mm 3 The mean tumor volume of the treated mice was 1817 mm 3 The mean tumor volume of the two treatment groups was 723 mm 3 The mean tumor volume of the three treated groups of mice was 452 mm 3 The mean tumor volume of the four treated groups of mice was 3639 mm 3 .

[0086] The results showed that the CAR-T cells induced by AntiCD8-LNP-TIM-3-2 designed in this application in mice had the best inhibitory effect on tumor growth in C57BL6 mice, indicating that the ScFv (TIM-3) antibody after sequence optimization had a more significant tumor killing effect. After injection of rituximab, CAR-T cells had basically no effect on tumors, indicating that rituximab can activate the suicide system RQR8 of CAR-T cells in the body, and cell activity is controlled by the suicide gene system.

Claims

1. A CD8 antibody-modified anti-TIM-3 lipid nanoparticle, characterized in that: The lipid nanoparticle contains a chimeric antigen receptor; the chimeric antigen receptor is obtained by sequentially connecting the following modules: a guide, a single-chain antibody TIM-3, a CD8 Hinge region, a CD28 transmembrane region, a CD28-4-1BB co-stimulatory region, a CD3ζ intracellular region, a self-cleavage region T2A, and a suicide gene; the artificial nucleic acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.2; The heavy chain amino acid sequence of the CD8 antibody is shown in SEQ ID NO.12, and the light chain amino acid sequence is shown in SEQ ID NO.13; The method for preparing the lipid nanoparticles comprises constructing a chimeric antigen receptor to obtain a recombinant plasmid, encapsulating it with LNP, and then coupling it with a CD8 antibody to obtain a CD8 antibody-modified anti-TIM-3 lipid nanoparticle; The composition of the LNP is: ionizable lipid DLin-MC3-DMA, cholesterol, DMG-PEG2000, DSPC, DSPE-PEG-maleimide, and the mass ratio is 2.18:1.00:0.27:0.54:0.

1.

2. Use of the lipid nanoparticles according to claim 1 in the preparation of anti-breast cancer drugs.

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